This invention relates generally to a method of focusing light for optical measurements into volumetric spaces and, more specifically, to an autofocus system for use with measurements in wells within microplates.
Numerous investigative assays use fluorescence to identify or enumerate a target of interest. Fluorescent detection has many applications in serology, cytology, microbiology, and histopathology. A chief advantage of using fluorescence is the low levels at which fluorescence can be detected, enabling highly sensitive tests. A second advantage with the use of fluorescence is that different fluorescent compounds have various different excitation and emission wavelengths. This allows for development of an assay of multiple targets in a single sample, with the assay for each target of interest associated with a different wavelength as a marker. An additional advantage is that fluorescence does not require the use of radioisotopes, resulting in reagents that are both safe to use and can be disposed of more easily.
One application of the use of fluorescent based assays is in the screening of compounds to identify potential pharmaceuticals. The process of drug discovery includes the screening of vast numbers of drug candidates made by combinatorial chemistry, requiring an extremely large number of assays. To simplify this process, assay procedures are often automated. Automation greatly increases screening throughput, allowing for more cost effective isolation of possible new drugs.
One method of automating the screening process involves introducing samples into a microplate well. These wells are often small, cylindrical receptacles arranged in rows in a rectangular array on a plastic sample plate. Commonly used microplates have 96, 384, or more wells per plate. Automated handling of these plates allows for higher throughput in screening samples in microplates.
Presently, assays using microplates and fluorescence detect emission in a two-dimensional reading of microplate wells. For example, U.S. Pat. No. 5,589,351 teaches a fluorescence analysis system that detects light transmitted from wells in a microplate. The light from a well is gathered and transferred to a reflector which sequentially directs light to a single detector. This system allows the sequential reading of rows of wells. The reading of wells is effected two dimensionally, with each well read as a unitary source of emitted light. This eliminates the ability of the assay to gather information on localized events within the well. U.S. Pat. No. 5,784,152 teaches another microplate reader that detects fluorescent emission. In this reader optical elements are included to enable tuneable detection to specific wavelengths. Detection is again effected in a two dimensional manner.
Three dimensional reading of microplate wells would allow more information to be gathered while using microplates. The targeted fluorescence often is localized on the bottom of microplate wells. The liquid in the microplate wells often contains additional unbound fluorescent reagents. In standard two-dimensional microplate fluorescent assays, detection of fluorescence on the bottom of a microplate well in a homogenous liquid is not possible. Fluorescent emission at the bottom of the well would be masked by the background fluorescence emitted from the unbound fluorescent compounds in the rest of the depth of the well. Simplified high throughput screens ideally would allow detection of the bottom layer in a well without removal of the unreacted fluorescent reagents.
To be able to detect fluorescence from the bottom layer of a microplate well requires the ability to automatically focus on a thin layer at the bottom of the well to excite fluorescent emission. This requires that the light source be able to focus on a 30 to 150 micron depth at the bottom of the microplate well. This focal depth would create a virtual capillary at the bottom of the well, a focal layer with the area of the well bottom but a depth of only 30 to 150 microns.
The geometry of microplates complicates attempts to focus on the bottom of microplate wells. A standard focal length would be possible if the plates were uniform to optical tolerance. However the location of the bottom surface of a microplate well is not uniform to 30 to 150 micron tolerances. To overcome this problem requires devising a method to precisely locate the bottom of a microplate well and focus on this location. In a high throughput system, this method must be rapid and accurate.
One known method of detecting a bottom layer in a microplate uses fluorescence detection to optically autofocus on a target layer within a microplate well. When the focal spot of laser light is below the well base top, minimal fluorescence will be detected. When the microplate is moved along the z axis of a microplate well (z axis is the longitudinal axis of the microplate well), the focal spot at some point begins to cross the well base top. The light from the focal spot will begin to excite fluorescent emission from some of the fluorescent compounds in the well as the focal spot enters the well. When the entire focal spot is above the well base top, a maximum fluorescent intensity is reached. FIG. 2 is a graph of the fluorescence intensity as the focal spot is moved into the well. The beginning of the maximum plateau of fluorescent intensity is a point where the location of the plate when the focal spot is entirely above the well base top. This has been used to refocus the beam within a microplate well. However, in practice this method has proved difficult to effect. The time required to use fluorescent graphing to determine focal spot placement within a well is not rapid enough for high throughput applications.
It is therefore an object of the invention to provide a method and apparatus capable of automatically focusing on a thin layer at the bottom of a microplate well. The focusing procedure should be rapid, accurate, and adaptable to fluorescence measurement in heterogeneous assays containing unbound fluorescent reagents. An additional object of the invention is to be able to use the focus method to determine the volume of liquid within the microplate well.
The above objects are achieved through a method and apparatus that uses a focused beam of light to optically sense a reference point on a microplate, such as the location of the top surface of the microplate well base, i.e. the surface defining the bottom of the well, and use this reference location to refocus the beam of light on a target within the microplate well that is positioned in a defined relation to the reference location. A scan of the target layer allows an assay of a thin layer within a microplate well without optical interference from non-target locations within the microplate well, particularly where fluorescence is used to identify target substances.
In a first embodiment, this method is achieved by focusing a beam of light toward the material interface occurring at an optically detectable reference surface on the underside of a microplate. The reference surface is then moved relative to the focal spot of the beam of light. As this movement occurs, specular reflection from the reference surface is collected and directed through a focal aperture onto a light detector where the intensity of the specular reflection is measured. A peak intensity will be measured when the focal spot of the beam of light is on the reference surface, allowing a maximal amount of light to be directed through a focal aperture and onto a detector. Once the location of the reference surface is known, this location can be used to relocate the beam of light onto a target layer in the microplate well if the target layer is in a known relation to the reference surface. Once the focal spot is relocated to the target layer, fluorescence is excited and detected using a scan of the target layer.
In an alternate embodiment the autofocus method, the focal aperture and detector are replaced with a position sensitive detector. Again a focal spot of a beam of light is directed to a reference surface on the underside of a microplate well. The reference surface is moved relative to the focal spot of the beam of light but the detector is kept stationary. As this movement occurs, the specular reflection from the reference standard is directed onto the position sensitive detector having a sensitive area large enough to image the reflected focal spot during its motion. The position sensitive detector measures both the intensity of the reflected light and the position of the reflected light on the detector. As in the first embodiment, the measurement of the maximum detected light intensity correlates to where focal spot is on the reference surface. When the light reaches the maximum intensity of measured specular reflection, the light will also be detected at a position on the position sensitive detector. This position on the position sensitive detector is the location when the focal spot is targeted onto the reference surfaces. Once this position on the position sensitive detector is known, it can be used as a deterministic indicator for finding the position of the reference surface at other locations on the microplate. As in the first embodiment, once the reference surface is located, it is used to reposition the focal spot onto a target layer which is subsequently optically scanned.